Rat Cornea Research Articles

Activation of the ATP-P2X pathway by TRPV4 in acute ocular hypertension

To measure the expression of transient receptor potential cation channel subfamily V member 4 (TRPV4) in the rat cornea and determine whether it is related to adenosine triphosphate (ATP) generation in a rat model of acute ocular hypertension (AOH). Immunofluorescence staining of TRPV4, P2X2 receptor, P2X3 receptor, and β3-tubulin in rat corneal longitudinal sections and paved was performed to clearly display histological structures. Rat models of AOH and agonist/antagonist-treated groups were established and corneal ATP was measured using an ATP assay. The independent t-test and simple linear correlation model were adopted for statistical analyses. Immunofluorescence staining of rat cornea sections revealed that epithelial and endothelial membranes showed strong immunoreactivity for TRPV4 and P2X2 receptor and coexpression with β3-tubulin in the rat corneal epithelial layer. Corneal ATP was significantly higher in the AOH rat model than in the control (P<0.05) and apparently lower after pretreatment by applying eyedrops of TRPV4 antagonist RN1734 with 30-40 mm Hg intraocular pressure (IOP; P<0.05). A simple linear regression model showed a positive correlation between rat corneal ATP and IOP values (R 2=0.996, P=0.0134) from the normal IOP (113 mm Hg) to 40 mm Hg. At 10-40min after anterior chamber injection of GSK1016790A (0.01 mL, 50 nmol/L in 0.9% NaCl), corneal ATP was significantly higher than in the control group (P<0.05), which peaked at 10min. The ATP concentration of the normal epithelium was higher than that of the endothelium in the AOH rat model and after anterior chamber injection of GSK1016790A (P<0.05). The ATP concentration in the AOH rat cornea is increased by TRPV4 activation.

Sirt1 attenuates diabetic keratopathy by regulating the endoplasmic reticulum stress pathway

AimsThe objectives of this study were to explore physiological and pathological changes in the corneas of diabetic rats by intervening in the expression of silent information regulator 1 (Sirt1) and to investigate whether Sirt1 can regulate the activation of endoplasmic reticulum stress (ERS) while influencing corneal epithelial cell apoptosis under high glucose conditions. Materials and methodsUsing 8-week old Sprague-Dawley rats, we established a model of type 1 diabetes, with or without Sirt1 intervention. Clinical evaluation was performed once per week. Primary rat corneal epithelial cells (RCECs) were cultured by combining Sirt1 intervention under high glucose conditions. Generation of reactive oxygen species (ROS), apoptosis, and the expression of Sirt1 and ERS-related proteins were evaluated in rat corneal tissues and RCECs. Key findingsDuring the intervention, clinical evaluation of the ocular surface, ROS generation, apoptosis, and protein expression of ERS-related proteins in corneal tissue and cultured RCECs were altered with Sirt1expression levels. SignificanceSirt1 expression influences the pathological progression of diabetic keratopathy, plays an important role in regulating the ERS pathway, and decreases corneal epithelial cell apoptosis.

Comparative behaviour of electrospun nanofibers fabricated from acid and alkaline hydrolysed gelatin: towards corneal tissue engineering

Gelatin is a hydrolysed form of collagen which is categorised as type A (acid hydrolysed) or type B (base hydrolysed), based on method of hydrolysis. In this work, we have studied the comparative properties of electrospun nanofibers of gelatin A (GA) and gelatin B (GB) prepared using a ternary solvent (glacial acetic acid/ethyl acetate/water). In addition, silk fibroin was used to improve the integrity and stability of gelatin nanofibrous sheets. Silk fibroin is known to serve as a potential implantable biomaterial because of its optimal mechanical stability, transparency, cell proliferation and tunable degradability. GA scaffold and silk permeated gelatin A nanofibrous scaffold (SFG) showed 50-70% of transparency comparable to that of adult rat and goat cornea. ATR-FTIR analysis showed the presence of native functional groups and bonds. Scanning electron microscope (SEM) micrographs revealed the porous nature of the scaffolds i.e., 48.08± 3.35 and 51.38 ± 3.72 for GA and GB, respectively. GB nanofibrous scaffolds got dissolved instantly in phosphate buffer solution (PBS), while GA scaffolds remained stable up to ∼ 11 h at 25˚C, however got degraded within 12 h when incubated at 37˚C. Furthermore, SFG scaffolds physically cross-linked with ethanol vapour displayed relatively improved stability when examined for at least 6 days at 37°C, minor weight loss when incubated in a lysozyme solution and facilitated the cellular proliferation significantly. Liquid retention capacity of the electrospun GA nanofibrous scaffolds was found to be nearly 800% from its original weight within 48 h. The electrospun nanofibrous scaffolds i.e., SFG facilitated ~72% of cellular proliferation compared to the control for SIRC [Statens Seruminstitut Rabbit Cornea] fibroblasts cells by 5th day of cell culture. Comparative outcomes reveal that the electrospun GA nanofibers possess significantly better integrity and noted stability in an aqueous environment compared to GB nanofibrous scaffold. Moreover, owing to the enhanced integrity and better stability the GA nanofibrous scaffold in combination with silk fibroin could be exploited in corneal tissue engineering applications where transparency plays an indispensable characteristic.

Depth-resolved Mueller matrix polarimetry microscopy of the rat cornea.

Mueller matrix polarimetry (MMP) is a promising linear imaging modality that can enable visualization and measurement of the polarization properties of the cornea. Although the distribution of corneal birefringence has been reported, depth resolved MMP imaging of the cornea has not been archived and remains challenging. In this work, we perform depth-resolved imaging of the cornea using an improved system that combines Mueller matrix reflectance and transmission microscopy together with nonlinear microscopy utilizing second harmonic generation (SHG) and two photon excitation fluorescence (TPEF). We show that TPEF can reveal corneal epithelial cellular network while SHG can highlight the presence of corneal stromal lamellae. We then demonstrate that, in confocal reflectance measurement, as depth increases from 0 to 80 μm both corneal depolarization and retardation increase. Furthermore, it is shown that the spatial distribution of corneal depolarization and retardation displays similar complexity in both reflectance (confocal and non-confocal) and transmission measurement, likely due to the strong degree of heterogeneity in the stromal lamellae.

Effect of Aging on Cornea of Male Albino Rat and the Possible Protective Role of Vitamin C

Background and Objectives: Cornea is a tissue in human eye that improves quality of image formed in retina. It is located in the anterior portion of eyeball. Aging is a biological phenomenon that involves increase of oxidative stress associated with gradual degradation of structure and function of cornea. Ascorbic acid (vitamin C) has been shown to have protective effects in repair of many corneal diseases. The aim of this study was to evaluate the histological and ultra-structural changes induced by aging on cornea of albino rat and the possible protective role of vitamin C. Materials and Methods: Fifty male albino rats were divided into 3 groups. Group I included 10 rats of 6 months old who received no medications. Group II included 20 rats of 18 months old and divided into two subgroups; IIA: rats received no medications and IIB rats received vitamin C. It was administered by gastric tube once daily (200 mg/kg body weight) for 36 days. Group III included 20 rats above 24 months old and divided into two subgroups; IIIA: rats received no medications, and IIIB rats received vitamin C of the same dose ,duration and route of administration as subgroup IIB. Histological, immune-histochemical, transmission electron microscopic and morphometric studies were performed.Results: Aging was associated with significant separation of collagen bundles, disorganized corneal stroma and cytoplasmic vacuolation with small dark nuclei of corneal epithelial cells. Vitamin C showed significant protection of these signs of aging.Conclusion: Aging affects the cornea of albino rats, and vitamin C has a protective effect on aging of cornea.

Effect of Ifosfamide on corneal genetic material and the role of lecithin and quercetin

The present study aimed to investigate the possible roles of lecithin and quercetin either singly or combined against Ifosfamide-induced DNA damage in corneas. Healthy female Rattus norvegicus rats weighing 180 ±10 gwere divided into seven groups eachcomprised of ten rats. Group I served as control and intraperitoneally injected with 0.25 mlof physiological saline (0.9% NaCl)for 5 days. Groups II and III wereorally administered lecithin at a dose of100 mg/kg body weight or quercetin at a dose of 50 mg/kgbody weight suspended indistilled water, respectively for six days; whereasgroup IV was used as positive control and intraperitoneally injected with Ifosfamide at a dose of 80mg/kg body weight for five days.Groups V and VI were injected with either quercetin orlecithin along with Ifosfamide by the same treatmentregimens, respectively. Group VII received a combination of both protective agents alongwith Ifosfamide and treated similarly. Corneas from all groups were used for comet assay. Results: The lecithin and quercetin treated groups were characterized by normal comet parameters compared to control. After Ifosfamide injection, all comet parameters were significantly increased (p˂0.05). No changes in comet parameters were observed for lecithin-Ifosfamide treated group as well as for quercetin-Ifosfamide one. Co-administration of lecithin and quercetin indicate significant increase (p˂0.05) in all comet parameters (group VII). All together led to conclude that Ifosfamide causes DNA damage of rat cornea due to its oxidative stress and, co-administration of lecithin and quercetin was contradicting each other while using these antioxidants separately has beneficial effects on corneal genetic material.

Comparison between the Effect of Hyaluronic Acid and Mesenchymal Stem Cell Therapy on Cornea of Adult Albino Rats after Exposure to Alkali Burn (Light and Electron Microscopic Study)

Background: Corneal epithelial injuries are common in both physician and veterinary ophthalmology that often causes extensive damage and results in permanent visual impairment. Aim of work: to evaluate the beneficial effect of hyaluronic acid and MSCs in treatment of experimental alkali corneal burn. Subjects and methods: thirty adult albino rats of both sexes. Ten rats were used to harvest BM-MSCs while the others were divided into four groups. Group I was the control group. Group II with unilateral alkali-burnt cornea these rats were sacrificed after 1 day and the other sacrificed after 2 weeks. Group III were rats with unilateral hyaluronic acid-treated alkali-burnt corneas for 2 weeks. Group IV were rats with MSCs treated alkali-burnt corneas. Immune histochemical staining for CD44 and vimentin was performed. The corneas were examined using light microscopic, transmission electron microscopic and morphometric studies. Results: Corneal alkali burn resulted in desquamation of corneal epithelium in group II. The epithelial cell layers had vacuolated cytoplasm with pyknotic nuclei. The stroma contained irregularly arranged collagen fibers with wide spacing and congested blood vessels with cellular infiltration. Groups III and IV showed improvement of the histological and electron microscopic changes described in group II. Conclusions: The use of MSCs in the acute stage of corneal chemical trauma was associated with faster recovery of the wounded cornea when compared with the effect of hyaluronic acid sodium salt.

PTEN Inhibition Accelerates Corneal Endothelial Wound Healing through Increased Endothelial Cell Division and Migration.

PurposeTo investigate the role of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) in the regulation of corneal endothelial cell (CECs) focusing on proliferation and migration, and to further evaluate the application of PTEN inhibitors in the treatment of corneal endothelial dysfunction in a rat model.MethodsExpression of PTEN in human and rat corneal endothelium was determined by immunocytochemistry, western blotting, and ELISA. A small molecular inhibitor of PTEN, bpV(pic), was applied in the culture of human CEC cell line B4G12 and organ-cultured rat cornea in the presence of transforming growth factor beta 2 (TGF-β2). Cell cycle status was detected by flow cytometry and BrdU staining. Subcellular localization for endogenous p27Kip1 was detected by immunocytochemistry and western blotting. Moreover, exogenous transfected YFP-p27Kip1 was observed under a fluorescent microscope. Cell migration was examined with a wound scratch model and transwell invasion assay. Finally, bpV(pic) was intracamerally injected in a rat corneal endothelial injury model. The wound healing process was evaluated by slit lamp biomicroscopy, optical coherence tomography, histological and scanning electron microscope examination.ResultsThe expression of PTEN in human corneal endothelium was higher compared with rat, which we speculate was mostly responsible for the relatively less proliferation capacity of human CEC than rat. PTEN inhibition by bpV(pic) could reverse TGF-β2-induced CEC G1-arrest by alleviating p27Kip1 nuclear accumulation and decreasing total p27Kip1 expression. In addition, bpV(pic) promoted CEC migration, which acted synergistically with TGF-β2. Finally, intracameral injection of bpV(pic) could promote corneal endothelial wound healing in a rat model.ConclusionsOur study provided experimental basis for the development of therapeutic agent targeting on PTEN for the treatment of corneal endothelial dysfunction.

Long-Term Exposure to Urban Particulate Matter on the Ocular Surface and the Incidence of Deleterious Changes in the Cornea, Conjunctiva and Retina in Rats.

We investigated the time-dependent deleterious ocular changes induced by urban particulate matter (UPM) in vitro and in vivo. UPM treatment decreased human corneal epithelial cell migration and survival. Fluorescein scores were consistently increased by UPM application for 16 weeks. One week of rest at 2 or 4 weeks led to a recovery trend, whereas two weeks of rest at 8 weeks induced no change. UPM treatment decreased the tear film break-up time at 2 weeks, which was thereafter maintained until 16 weeks. No changes were found after periods of rest. UPM-treated eyes exhibited greater corneal epithelium thickness than normal eyes at 2 weeks, which recovered to normal at 4 and 8 weeks and was significantly decreased at 16 weeks. Apoptotic cell number in the epithelium was increased at 2 weeks, which remained constant except at 8 weeks. IL-6 expression in the cornea of the right eye continually increased for 16 weeks, and significant recovery was only observed at 8 weeks after 2 weeks of rest. Ocular pressure was significantly increased in the right eye at 12 and 16 weeks. Topical UPM application to the eye induced deleterious changes to various closely related parts of the eye.

Transcriptomic profiling of human corneal epithelial cells exposed to airborne fine particulate matter (PM2.5)

PurposeTo explore the molecular mechanisms of PM2.5-induced dysfunction in human corneal epithelial cells (HCECs) and the potential role of the plasminogen activator inhibitor type-2 (PAI-2) in PM2.5-induced autophagy in vitro and in vivo. MethodsRNA-Seq was performed to identify the differentially expressed genes (DEGs) in PM2.5-exposed HCECs compared to unexposed condition, followed by validation via real-time PCR (qRT-PCR). Corneal fluorescein staining and tear secretion were assessed in the PM2.5-exposed rat model. The expression of PAI-2 and autophagy-related markers were examined via immunoblotting, immunofluorescence staining and/or qRT-PCR in PM2.5-exposed or unexposed HCECs and rat corneas. PAI-2-knockdown HCECs were generated to study PAI-2's role in the PM2.5-induced autophagy in HCECs. ResultsA total of 434 DEGs—240 up-regulated and 194 down-regulated—were identified in PM2.5-exposed HCECs rather than unexposed HCECs. The expression of a few genes related to proliferation, inflammation, and aryl hydrocarbon stimulation were significantly altered by PM2.5 exposure. PAI-2 expression was up-regulated in PM2.5-exposed HCECs, sharing a similar fluctuation trend with autophagy-related markers LC3B II and BECN1 according to various exposure periods. Moreover, PAI-2 knockdown significantly suppressed the expression of LC3B and BECN1 in PM2.5-exposed HCECs. The corneal fluorescein staining was enhanced and tear secretion was significantly reduced in PM2.5-exposed rat eyes. PAI-2 expression was also increased in PM2.5-exposed rat corneas, together with the up-regulation of several autophagy-related markers. ConclusionThe present study identified the altered expression of hundreds of genes in PM2.5-exposed HCECs, which suggests the importance of PM2.5 for cornea health. The involvement of PAI-2 was discovered in the PM2.5-induced autophagy in HCECs as well as likely in rat corneas, which implied that PAI-2 may become a potential target of clinical treatment of PM2.5-associated ocular surface diseases.

PPARγ: the dominant regulator among PPARs in dry eye lacrimal gland and diabetic lacrimal gland.

To investigate the regulatory roles of the members of the peroxisome proliferator-activated receptor (PPAR) family in lacrimal gland dysfunction under conditions of desiccating stress or diabetes. Quantitative polymerase chain reaction (qPCR) was used to examine the expression of PPARs in the cornea, conjunctiva, meibomian gland, and lacrimal gland in adult rats. The rats were divided into 3 groups: a control group, dry eye group, and diabetic group. The phenol red threads test, tear film break-up time (BUT) test and fluorescein staining were carried out to evaluate the development of dry eye. Based on bioinformatics research, qPCR was used to examine the expression level of PPARγ, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), sirtuin 1 (Sirt1), myeloid differentiation factor 88 (MyD88) and transforming growth factor-β (TGF-β) in the lacrimal glands. PPARα and PPARβ/δ were mainly expressed in the conjunctiva and the lacrimal gland, respectively. However, PPARγ was expressed in both the conjunctiva and lacrimal gland, at much higher levels than those measured for PPARα and PPARβ/δ. Dry eye rats and diabetic rats both showed decreased tear secretion, shortened BUT, and increased corneal staining. Significant changes in gene expression were observed compared with the control group. In the lacrimal glands of dry eye rats and diabetic rats, expression of PPARγ decreased (P<0.05), expression of Sirt1 also decreased (P<0.01), whereas expression of TNF-α, IL-1β, IL-6, MyD88, and TGF-β increased (P<0.05). Among PPARs, PPARγ might play a dominant role in the regulation of metabolic- and inflammatory-signaling pathways on the ocular surfaces and in lacrimal glands. Down-regulation of PPARγ is highly relevant to lacrimal gland dysfunction under desiccating-stress and diabetic conditions. PPARγ, thus, is a potential therapeutic target in the treatment of environment- or diabetes-induced dry eye diseases.

Nanosensitive optical coherence tomography to assess wound healing within the cornea.

Optical coherence tomography (OCT) is a non-invasive depth resolved optical imaging modality, that enables high resolution, cross-sectional imaging in biological tissues and materials at clinically relevant depths. Though OCT offers high resolution imaging, the best ultra-high-resolution OCT systems are limited to imaging structural changes with a resolution of one micron on a single B-scan within very limited depth. Nanosensitive OCT (nsOCT) is a recently developed technique that is capable of providing enhanced sensitivity of OCT to structural changes. Improving the sensitivity of OCT to detect structural changes at the nanoscale level, to a depth typical for conventional OCT, could potentially improve the diagnostic capability of OCT in medical applications. In this paper, we demonstrate the capability of nsOCT to detect structural changes deep in the rat cornea following superficial corneal injury.

Protective Effect of Vitamin C against Infancy Rat Corneal Injury Caused by Acute UVB Irradiation.

Purpose Studies have shown that corneas of young children were more susceptible to Ultraviolet B (UVB) radiation damage. However, there exist limited information about the harm of UVB to eyes and preventive measures on infancy. Vitamin C as an antioxidant is widely used to prevent many diseases. Therefore, the aim of this study was to explore the protective effect of vitamin C on the cornea of infant rats with acute UVB injury. Method Thirty-six infant rats were randomly divided into three groups: control (CON) group, UVB (UVB) group, and UVB+vitamin C (UVB+VitC) group. The UVB group was exposed to UVB irradiation (8 J/cm2, 15 min/d, 7 d) and the UVB+vitamin C group suffered the same UVB irradiation treated with vitamin C at the dose of 40 mg/kg via intraperitoneal injection. Then, corneal morphology was detected in vivo and in vitro at 7 d post-UVB exposure. Furthermore, serum inflammatory factors (IL-1, IL-6, and TNF-α) and oxidative status (4-HNE and MDA) were detected by ELISA, and the expression of vascular endothelial growth factor-α (VEGF-α) and superoxide dismutase (SOD) in the cornea was detected by western blot or immunofluorescent staining. Results Slit lamp detection revealed that the area of corneal desquamation and corneal neovascularization in the UVB+VitC group was significantly less than those in the UVB group at 7 d post-UVB exposure (all p < 0.05). OCT results showed that the thickness of the central cornea in the UVB+VitC group was decreased than that in the UVB group (p < 0.05). The serum inflammatory factors (IL-1, IL-6, and TNF-α) and oxidative status (4-HNE and MDA) in the UVB group were significantly increased compared with the CON group (all p < 0.05), while those factors in the UVB+VitC group were decreased compared with those in the UVB group. Furthermore, the expression of VEGF-α in the UVB+VitC group was dramatically decreased compared with that in the UVB group (p < 0.05), and the expression of SOD2 in the UVB+VitC group was dramatically increased compared with that in the UVB group at 7 d post-UVB exposure (p < 0.05). Conclusion Vitamin C could protect infant rats from corneal injury induced by UVB via alleviating corneal edema, improving corneal inflammatory reaction, and decreasing VEGF-α expression.

Design of ocular drug delivery platforms and in vitro - in vivo evaluation of riboflavin to the cornea by non-interventional (epi-on) technique for keratoconus treatment

AimKeratoconus is a common and progressive eye disease characterized by thinning and tapering of the cornea. This degenerative eye disease is currently treated in the clinic with an interventional technique (“epi-off”) that can cause serious side effects as a result of the surgical procedure. The aim of this project is to design innovative formulations for the development of a riboflavin-containing medicinal product to develop a non-invasive (“epi-on”) keratoconus treatment as an alternative to current treatment modalities. MethodsNanostructured lipid carriers (NLCs) were successfully loaded with either riboflavin base of riboflavin-5-phosphate sodium and designed with either Stearylamine (positive charge) or Trancutol P (permeation enhancer). In vitro characterization studies, cytotoxicity and permeability studies were performed. Selected formulations and commercial preparations were applied and compared in ex-vivo corneal drug accumulation and transition studies. Furthermore, in vivo studies were performed to assess drug accumulation in the rat cornea and the corneal stability after NLC treatment was investigated via a biomechanical study on isolated rabbit corneas. ResultsBoth in vitro and ex-vivo as well as in vivo data showed that from the prepared NLC formulations, the most effective formulation was riboflavin-5-phosphate sodium containing NLC with Transcutol P as permeation enhancer. It possessed the highest drug loading content, low accumulation in the cornea but high permeability through the cornea as well as the highest functional performance in corneal crosslinking. ConclusionTopical application of riboflavin-5-phosphate sodium loaded NLC systems designed with permeation enhancer Transcutol P may act as a potential alternative for non-invasive keratoconus treatments.

A Novel CD147 Inhibitor, SP-8356, Attenuates Pathological Fibrosis in Alkali-Burned Rat Cornea.

The corneal fibrotic responses to corneal damage often lead to severe corneal opacification thereby resulting in severe visual impairment or even blindness. The persistence of corneal opacity depends heavily on the activity of corneal myofibroblast. Myofibroblasts are opaque and synthesize a disorganized extracellular matrix (ECM) and thus promoting opacification. Cluster of differentiation 147 (CD147), a member of the immunoglobulin superfamily, is known to play important roles in the differentiation process from fibroblast to myofibroblast in damaged cornea and may therefore be an effective target for treatment of corneal opacity. Here, we examined the therapeutic efficacy of novel CD147 inhibiting verbenone derivative SP-8356 ((1S,5R)-4-(3,4-dihydroxy-5-methoxystyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one) on corneal fibrosis. Topical SP-8356 significantly reduced corneal haze and fibrosis in the alkali-burned cornea. In detail, SP-8356 inhibited both alpha-smooth muscle actin (α-SMA) expressing myofibroblast and its ECM-related products, such as matrix-metalloproteinase-9 and collagen type III and IV. Similar to SP-8356, topical corticosteroid (prednisolone acetate, PA) also reduced the ECM-related products and opacification. However, prednisolone acetate failed to decrease the population of α-SMA-positive corneal myofibroblast. In conclusion, SP-8356 is capable enough to prevent corneal haze by preventing pathological fibrosis after severe corneal damage. Therefore, SP-8356 could be a potentially promising therapeutic drug for corneal fibrosis.

Pathogenic Role of PPARα Downregulation in Corneal Nerve Degeneration and Impaired Corneal Sensitivity in Diabetes.

The purpose of this study was to investigate the protective role of peroxisome proliferator–activated receptor α (PPARα) against diabetic keratopathy and corneal neuropathy. Corneal samples were obtained from human donors with and without diabetes. Streptozotocin-induced diabetic rats and mice were orally treated with PPARα agonist fenofibrate. As shown by immunohistochemistry and Western blotting, PPARα was downregulated in the corneas of humans with diabetes and diabetic rats. Immunostaining of β-III tubulin demonstrated that corneal nerve fiber metrics were decreased significantly in diabetic rats and mice, which were partially prevented by fenofibrate treatment. As evaluated using a Cochet-Bonnet aesthesiometer, corneal sensitivity was significantly decreased in diabetic mice, which was prevented by fenofibrate. PPARα−/− mice displayed progressive decreases in the corneal nerve fiber density. Consistently, corneal sensitivity was decreased in PPARα−/− mice relative to wild-type mice by 21 months of age. Diabetic mice showed increased incidence of spontaneous corneal epithelial lesion, which was prevented by fenofibrate while exacerbated by PPARα knockout. Western blot analysis revealed significantly altered neurotrophic factor levels in diabetic rat corneas, which were partially restored by fenofibrate treatment. These results indicate that PPARα protects the corneal nerve from degeneration induced by diabetes, and PPARα agonists have therapeutic potential in the treatment of diabetic keratopathy.

Effect of Aging on the Cornea of Male Albino Rat and Possible Therapeutic Role of Royal Jelly

Abstract Introduction Nowadays interest in aging has greatly increased, Aging is a complex natural process involving every molecule, cell, and organ in the body that is associated with tissue dysfunction in many organs. Aging of the cornea causes major eye effects and leads to substantial cost in medical and social terms. These effects include the highly prevalent dry eye disease (DED) that affects both visual function and quality of life in elderly. Symptoms of (DED) include, ocular pain, visual disturbances, and increase lacrimation. Functional foods such as Royal jelly (RJ) have a growing attention because of consumers increasing concerns about their health. Its importance not only for its nutritional properties but also for its functional and biological properties such as antioxidant, anti-inflammatory, antibacterial, antiviral, and anti-ulcerous activities. It is used as a cheap natural source in daily life and medicine. (RJ) is a complex substance composed of proteins, sugars, lipids, amino acids, vitamins, and minerals. Aim The present study aimed to investigate the histological effect of aging on the cornea of male albino rat and possible therapeutic role of (RJ) on senile group. Materials and Methods Twenty-four male albino rats were used in this study divided into Group I: consisted of 6 adult male rats aged 3- 6 months. Group II: consisted of 18 senile male rats aged 18-24 months, were further subdivided into three subgroups as follows: Group II A: (n = 6) negative control senile rats, not subjected to any procedure for 4 weeks. Group II B: (n = 6) control senile rats and were given distilled water by oral gavage once daily for 4 weeks. Group II C: (n = 6) senile rats were given (RJ) by oral gavage dissolved in distilled water once daily for 4 weeks. At the end of the experiment, rats were sacrificed after being deeply anesthetized with ether according to the protocol of the Committee of Animal Research Ethics (CARE). The cornea of each animal was carefully dissected out after death and immediately fixed in 10% formalin for preparation of paraffin blocks 5 micrometer thickness. Sections were stained with hematoxylin and eosin (I-I&amp;E), Masson's trichrome and periodic acid-Schiff (PAS). Statistical analysis and quantitative morphometric study were done. Results Light microscopic examination of corneas of senile rats revealed different pathological changes included irregularity in the surface epithelium as well as surface erosions and cytoplasmic vacuolations. The stroma showed widely separated collagen fibers with decreased keratocyte density. It was concluded that (RJ) supplementation to senile rats obviously unproved all layers of the cornea histologically.

Experimental keratitis in rats caused by Acanthamoeba griffini: A kinetic histopathological study.

The aim of this study was to evaluate the inflammation process that resulted from the inoculation of Wistar Rats with Acanthamoeba griffini, a virulent T3 Acanthamoeba genotype that produces keratitis. Haematoxylin and eosin, periodic acid stain, immunohistochemistry and morphometry were used to analyse tissues from rats of an Acanthamoeba keratitis (AK) model. Two weeks after inoculating the rats with Agriffini trophozoites, the thickness of the stroma had diminished, followed by an increase in thickness at 4weeks. At the latter time, an abundance of inflammatory infiltrate cells was observed, some found to express IL-1β, IL-10 and/or caspase 3. Intercellular adhesion molecule-1 was expressed in corneal blood vessels amid the abundant vascularization characteristic of the development of AK. Through an immunohistochemical technique, trophozoites were detected at 2 and 4weeks post-inoculation. By 8weeks, there were a low number of trophozoites and cysts and the corneas of infected rats were similar in thickness to those of the controls. Thus, the rats were capable of healing experimental AK in the present rat model. Diverse immunological mechanisms regulated the inflammatory process in acute AK induced by Agriffini in a murine model.

Real-time physiological measurements of oxygen using a non-invasive self-referencing optical fiber microsensor.

Reactive molecular oxygen (O2) plays important roles in bioenergetics and metabolism and is implicated in biochemical pathways underlying angiogenesis, fertilization, wound healing and regeneration. Here we describe how to use the scanning micro-optrode technique (SMOT) to measure extracellular fluxes of dissolved O2. The self-referencing O2-specific micro-optrode (also termed micro-optode and optical fiber microsensor) is a tapered optical fiber with an O2-sensitive fluorophore coated onto the tip. The O2 concentration is quantified by fluorescence quenching of the fluorophore emission upon excitation with blue-green light. The micro-optrode presents high spatial and temporal resolutions with improved signal-to-noise ratio (in the picomole range). In this protocol, we provide step-by-step instructions for micro-optrode calibration, validation, example applications and data analysis. We describe how to use the technique for cells (Xenopus oocyte), tissues (Xenopus epithelium and rat cornea), organs (Xenopus gills and mouse skin) and appendages (Xenopus tail), and provide recommendations on how to adapt the approach to different model systems. The basic, user-friendly system presented here can be readily installed to reliably and accurately measure physiological O2 fluxes in a wide spectrum of biological models and physiological responses. The full protocol can be performed in ~4 h.

Prolonged Duration Topical Corneal Anesthesia With the Cationic Lidocaine Derivative QX-314

PurposeTopical corneal local anesthetics are short acting and may impair corneal healing. In this study we compared corneal anesthesia and toxicity of topically applied N-ethyl lidocaine (QX-314) versus the conventional local anesthetic, proparacaine (PPC).MethodsVarious concentrations of QX-314 and 15 mM (0.5%) PPC were topically applied to rat corneas. Corneal anesthesia was assessed with a Cochet-Bonnet esthesiometer at predetermined time points. PC12 cells were exposed to the same solutions to assess cytotoxicity. Repeated topical corneal administration in rats was then used to assess for histologic evidence of toxicity. Finally, we created uniform corneal epithelial defects in rats and assessed the effect of repeated administration of these compounds on the defect healing rate.ResultsQX-314 (15 mM) and PPC (15 mM) caused similar total duration (114 ± 17 and 87 ± 16 minutes, respectively; P = 0.06) of anesthesia. The depth of anesthesia was similar between these low-dose groups at 15 minutes after application (1.8 ± 0.3- and 2.0 ± 0.8-cm filament lengths). QX-314 (100 mM) provided more prolonged corneal anesthesia (174 ± 13 minutes; P < 0.0001), with improved depth at 15 minutes (0.7 ± 0.3-cm filament length; P = 0.007). All tested concentrations of QX-314 demonstrated similar or less toxicity than 0.5% PPC.ConclusionsTopical administration of QX-314 is effective for corneal anesthesia and demonstrates no histologic signs of local toxicity in a rodent model. In higher concentrations, QX-314 provides more than twofold the duration of anesthetic effect than does 0.5% PPC.Translational RelevanceOur study reveals a clinically relevant compound providing prolonged duration topical corneal anesthesia.